When reactive substances such as protein, peptide and amino acid having an amino group(s) coexist with a reducing sugar such as aldose having an aldehyde group(s), they combine nonenzymatically and irreversibly through the amino and aldehyde groups, which is followed by amadori rearrangement to form an amadori compound. The production rate of an amadori compound being a function of concentration of reactants, period of contact, temperature and the like, various useful information can be derived from the amount of amadori compounds in a sample containing reactive substance(s). Examples of materials containing an amadori compound include food products such as soy sauce and body fluids such as blood.
In a living body, fructosylamines are formed through the reaction between glucose and an amino acid, and the resultant glycated derivatives of hemoglobin, albumin and proteins in blood are called glycohemoglobin, glycoalbumin and fructosamine, respectively. As the concentration of these glycated derivatives in blood reflects an average of blood sugar levels over a particular period of time, it can be a significant index for diagnosis and control of conditions of diabetes. Therefore, the establishment of a method of measuring an amadori compound in blood is clinically useful.
Further, a state of preservation and period after production of a food product can be estimated on the basis of the amount of amadori compounds in the food product. Therefore, the method of measuring an amadori compound can also contribute to the quality control of a food product.
Thus, an assay of amadori compounds should be useful in wide fields involving medicine and food products.
As assay of amadori compounds, there has been known method which utilizes high performance liquid chromatography [Chromatogr. Sci. 10: 659 (1979)], a column filled with solid materials to which boric acid is attached [Clin. Chem. 28: 2088 (1982)], electrophoresis [Clin. Chem. 26:1598 (1980)] or antigen-antibody reaction [JJCLA 18: 620 (1993), J. Clin. Lab. Inst. Reag. 16:33-37 (1993)], a method for measuring the amount of fructosamine [Clin. Chem. Acta 127:87-95 (1982)], a calorimetric determination following the oxidization with thiobarbituric acid [Clin. Chem. Acta 112:179-204 (1981)], or the like. The existing methods, however, require an expensive device(s) and are not necessarily accurate and rapid enough.
In the field of clinical assay and food analysis, a method utilizing enzymatic process has recently been widely used because, owing to characteristics of enzymes (specificity in terms of substrate, reaction, structure, active site, etc.), an intended substance can be selectively analyzed with accuracy and rapidity.
There have already been proposed assays which comprise reacting an oxidoreductase with amadori compounds and determining oxygen consumption or hydrogen peroxide generation as an index of the amount of amadori compounds (e.g. Japanese Patent Publication (KOKOKU) Nos. 5-33997 and 6-65300, and Japanese Laid-Open Patent Publication Nos. 2-195900, 3-155780, 4-4874, 5-192293 and 6-46846). Further, assays of glycated protein for the diagnosis of diabetes have been disclosed (Japanese Laid-Open Patent Publication Nos. 2-195899, 2-195900, 5-192193 and 6-46846).
The decomposition of amadori compounds catalyzed by an oxidoreductase can be represented by the following reaction scheme: EQU R--CO--CH.sub.2 --NH--R.sup.2 +O.sub.2 +H.sub.2 O.fwdarw.R.sup.1 --CO--CHO+R.sup.3 --NH+H.sub.2 O.sub.2
wherein R.sup.1 is an aldose residue and R.sup.2 is an amino acid, protein or peptide residue.
Examples of enzymes which catalyze the above reaction include fructosyl amino acid oxidase derived from microorganisms [e.g., strains of Corynebacterium (Japanese Patent Publication Nos. 5-33997 and 6-65300), strains of Asperqillus (Japanese Laid-Open Patent Publication No. 3-155780), etc.], ketoamine oxidase derived from microorganisms [e.g., strains of Corynebacterium, Fusarium, Acremonium or Debaryomyces (Japanese Laid-Open Patent Publication No. 5-192193)], fructosylamine deglycase [e.g., strains of Candida (Japanese Laid-Open Patent Publication No. 6-46846)], alkyllysinase which can be prepared according to the method described in J. Biol. Chem., Vol. 239, pp. 3790-3796 (1964), and the like.
Assays involving these existing enzymes, however, had some drawbacks.
For instance, an amadori compound in blood which serves as an index for the diagnosis of diabetes is a glycated protein normally formed when a glucose is bound to a lysine residue at its .di-elect cons.-position on protein molecule [J. Biol. Chem. 26: 13542-13545 (1986)]. Therefore, it is necessary to use an enzyme highly specific to fructosyl lysine to conduct the determination of glycated proteins efficiently. However, the existing enzyme derived from Corynebacterium does not act on fructosyl lysine. Although an enzyme from Asperqillus acts on fructosyl lysine, it is less reactive with fructosyl lysine compared to other amadori compounds (see Table 5 below). Further, the action on the glycated protein or hydrolysis thereof is still unclear. Although the ketoamine oxidase described in Japanese Laid-Open Patent Publication No. 5-192193 is specific to glycated .alpha.-amine group on fructosyl valine, it cannot afford an accurate assay of glycated proteins where a lysine residue is bound to a sugar. Because the fructosylamine deglycase is highly specific to difructosyl lysine, it is not available in an assay specific to a substance having lysine residue which is glycated at the .di-elect cons.-position.
Furthermore, a method using an alkyllysinase cannot be reliable and accurate because said enzyme lacks in specificity and reacts with substances whose lysine residue is not bonded to only a sugar.
As described above, existing enzymes cannot necessarily give an accurate assay of glycated proteins and therefore the development of an enzyme highly specific to fructosyl lysine has been demanded.
In general, for the improvement of accuracy and usefulness of an assay involving an enzymatic process, it is essential to use an enzyme having a catalytic activity suitable for purposes of the assay. Thus, it is necessary to select an appropriate enzyme taking many factors such as a substance to be determined, as a substrate of the enzyme, the condition of a sample, measuring conditions and the like into consideration in order to carry out the assay with accuracy and reproducibility. To select a suitable enzyme, many enzymes must be previously obtained and characterized regarding the activity, substrate specificity, temperature stability, pH stability and the like. Therefore, it is necessary to develop more and more fructosyl amino acid oxidases and characterize the same.